Solid phase bioaffinity assays are based on the adsorption of antibodies or other biomolecules on a solid support, e.g., on the outer surface of spherical particles, or the inner surface of test tubes, microtitration wells or planar “chip” surfaces, which is then used to immobilize a second capture molecule or an analyte to be detected.
Today, many different coating techniques are known to produce a functionally active surface for solid phase applications. However, current expectations for assay sensitivity, kinetics and robustness set new demands for critical assay components such as the solid phase properties. Existing patterned surfaces are not always able to address the requirements for assay performance and higher surface binding capacity.
The simplest method to produce a solid phase coating is the adsorption of an unmodified protein onto a solid support, which in turn is usually made of polystyrene, polypropylene or some other plastic material or glass. The protein is dissolved in a buffer, e.g., carbonate or phosphate buffer, and applied to the surface of the solid support. The coating is performed in a humid environment, followed by a saturation step to block the unspecific binding sites and finally the surface is dried. However, because usually only a small fraction of the native protein retains its activity after such passive adsorption, the solid phase coating obtained in this way is not always the best choice in terms of binding capacity.
Therefore, several studies have sought to improve adsorption properties and the surface stability of protein coatings, e.g., when the protein is coupled to different “carrier” proteins prior to adsorption to the solid support. Modifications of the native structure of a macromolecule are also often desired to produce improved adsorption properties for demanding assays.
One such macromolecule is streptavidin, which is widely used to capture biotinylated proteins onto a solid phase. The ultimate nature of streptavidin to tightly bind biotin has been utilized in many solid phase-based applications, including microarrays, biosensors and other bioaffinity assays.
U.S. Pat. No. 5,061,640 describes the use of a larger precursor protein to enhance the adsorption of streptavidin. The method provides a coating process which improves the adhesion of the streptavidin to the solid support by conjugating streptavidin to a more hydrophobic carrier molecule like bovine serum albumin prior to coating on a hydrophobic solid support, thus leading to a solid phase with improved binding properties.
Another process to improve the adsorption properties of the coated substances is described in U.S. Pat. No. 6,638,728 B1, which describes the polymerization of monomeric streptavidin with bi-functional linkers to provide a mixture of dimers, trimers and tetramers of streptavidin, which have improved biotin binding capacity compared to a native streptavidin surface when adsorbed onto polystyrene.
The polymerization of thiolated streptavidin through disulphide groups has been shown to provide improvement in the solid phase properties as well, leading to enhanced surface stability by minimizing protein leakage from the solid phase during incubation when coated on polystyrene surfaces (Valimaa, L., Laurikainen, K., 2006: “Comparison-study of streptavidin coated microtitration plates”, J. Immunol. Methods 308, 203). This study, however, also revealed that although coatings based on thiolated streptavidin showed good solid phase properties and improved binding capacities compared to native streptavidin, other coatings showed higher adsorption capacities. Such coatings, on the other hand, tend to be unstable.
In fact, the solid phase properties are as well highly dependent on the coating conditions used, which in turn includes the buffer composition applied and the coating technique used. Thus, several studies have revealed the importance of the buffer additives in the printing solution to improve spot morphology and reproducibility. In order to reduce the variation between spot morphology and to improve the overall reproducibility of spot production, different surfactants are commonly used.
Thus, with US 2006/0223074 A1, spotting solutions for coating of a solid support comprising an alkylene diol, betaine, a detergent and a salt have been proposed. Betaine (N,N,N-trimethylglycine) has a zwitterionic structure, whereas the detergents disclosed in US 2006/0223074 are ionic or nonionic. Such compositions provide for high spot consistency and stability as well as ease of production and long-term storage.
Several other studies report optimized buffer composition on DNA-microarrays. However, although such studies reported improvement in signal intensity when betaine was used as an additive in the spotting solution, compared to normal spotting solution without additives, rather small total binding capacities were obtained with such coatings.
Thus there still exists a need to provide solid phase coatings for solid supports, wherein the solid phase displays high binding capacities while not compromising the stability properties.
Therefore it is an object of the present invention to provide such high capacity solid phases which may be used for highly efficient applications like microarrays, biosensors and other bioaffinity assays.